DNA purification
The DNA purification (also DNA preparation, DNA isolation ) describes the separation of DNA from a mixture or a solution containing a plurality of biomolecules.
principle
The purification of DNA can be achieved by various methods, which can also be combined with one another. The DNA purification can be carried out by using different purification methods , the effectiveness (the meaningful sequence) and the efficiency (the degree of purification) being monitored and quantified with analytical methods. In selecting the methods according to the chain length between genomic DNA, plastid - DNA , plasmids and viral DNA distinguished.
Tissues are occasionally shredded mechanically ( blender ) or enzymatically ( proteinase K ) before cell disruption. In electrophoresis and chromatography, a sample must first be filtered or centrifuged after cell disruption , as the larger fragments can clog the apparatus. To inactivate nucleases , EDTA or other chelators are usually added and work is carried out quickly at 4 ° C, e.g. B. with TE buffer and reaction vessels in an ice bath.
Properties of DNA
Due to the phosphoribose backbone, DNA has negative charges proportional to its chain length and, due to its relatively high molar mass, is insoluble in an acidic aqueous environment, since the phosphate groups are saturated with protons and the hydrate shell and thus the solubility are reduced as a result. DNA is also insoluble in a non-polar environment ( organic solvent ) due to the reduced hydrate shell and lower solubility. Because of the similar properties of RNA, the methods of RNA purification are similar to those of DNA purification.
Compared to intracellular proteins, DNA has a significantly higher molar mass, a higher density and no positive charges on the phosphoribose backbone. Due to the conjugated double bonds in the nucleobases , DNA absorbs ultraviolet light at a wavelength of 260 nm, which is used for photometric quantification. This allows the cleaning factors to be determined. An absorbance of 1 of a purified DNA solution corresponds to a concentration of 50 micrograms per milliliter for double-stranded DNA , 40 micrograms per milliliter for single-stranded DNA or RNA and 20 micrograms per milliliter for single-stranded oligonucleotides .
coloring
DNA can be made visible by various staining methods. As dyes and processes, for. B. Methylene blue , Stains-all or the silver coloring are used. Fluorescent dyes are e.g. B. 4 ', 6-diamidine-2-phenylindole , bisbenzimides such as Hoechst 33342 or phenanthridines such as acridine orange , ethidium bromide , propidium iodide , Gel Red or Gel Green . Other DNA-binding molecules are e.g. B. spermine , spermidine , polyethyleneimine , pentamidine and lexitropsine and DNA-binding proteins .
Separation process
DNA extraction
A series of extractions and precipitations is probably the most commonly used procedure.
Chromatography
DNA can be separated by size exclusion chromatography (SEC) based on its hydrodynamic volume . DNA can also be separated by anion exchange chromatography .
sedimentation
By density gradient centrifugation in a cesium chloride gradient, DNA can be separated based on its sedimentation constant .
With pulldown assays such as chromatin immunoprecipitation , DNA molecules are adsorbed on a matrix based on their affinity and isolated based on the properties of the matrix.
Electrophoresis
After another previous purification by agarose gel electrophoresis or by capillary electrophoresis, the DNA can be separated according to its electrical charge and hydrodynamic volume, both of which depend on the chain length and thus on the molar mass.
Filtration
In a series of microfiltration and several ultrafiltration , samples are also separated according to their hydrodynamic volume.
quantification
DNA can be quantified by photometry or by QPCR .
literature
- Friedrich Lottspeich , Haralabos Zorbas: Bioanalytics . Spektrum Akademischer Verlag, Heidelberg 1998, ISBN 978-3-8274-0041-3 .
- Cornel Mülhardt: The Experimenter: Molecular Biology / Genomics. Sixth edition. Spectrum Akademischer Verlag, Heidelberg 2008, ISBN 3-8274-2036-9 .
- J. Sambrook , T. Maniatis , DW Russel: Molecular cloning: a laboratory manual. 3rd edition (2001), Cold Spring Harbor Laboratory Press. ISBN 0-87969-577-3 .